Alkaline protease, method for its production,and detergent composition

ABSTRACT

ALKALINE PROTEASE IS OBTAINED BY PROPAGATING THE OR02GANISM BACILLUS LICHENIFORMIS A.T.C.C. NO. 21424 IN NUTRIENT MEDIA AND THEREAFTER RECOVERING THE ENZYME FROM THE MEDIA.

United States Patent 3,748,233 ALKALINE PROTEASE, METHOD FOR ITS PRO- DUCTION, AND DETERGENT COMPOSITION John P. Viccaro, Jamaica, N.Y., assignor to Lever Brothers Company, New York, N.Y. No Drawing. Filed Sept. 8, 1969, Ser. No. 856,182 Int. Cl. C12d 13/10 US. Cl. 195-66 R 6 Claims ABSTRACT OF THE DISCLOSURE Alkaline protease is obtained by propagating the organism Bacillus licheniformis A.T.C.C. No. 21424 in nutrient media and thereafter recovering the enzyme from the media.

The present invention relates to alkaline protease obtained as an elaboration product of the growth of the microorganism Bacillus licheniformis A.T.C.C. No. 21424 in a nutrient medium, to a method of producing the alkaline protease, and to detergent compositions containing the protease.

Alkaline protease is a term used to describe a proteolytic enzyme which exhibits optimum activity in an alkaline medium. Proteolytic enzymes of this nature are useful as an active ingredient in present day enzyme detergents and laundry presoak compositions. The enzyme is used for its ability to attack and remove the common soils and stains of clothing that have a significant protein content such as blood and grass stains.

A number of sources of alkaline protease are presently known. Some of the more important processes now in commercial use rely upon the fermentation of Bacillus subtilis and Bacillus licheniformis or some of the molds, such as the Aspergilli.

The applicant has found a strain of Bacillus licheniformis, A.T.C.C. No. 21424, which provides high yields of alkaline protease. Exceptional results are obtained when the organism is grown in media of certain characteristics. As a result, the enzyme provided by the present invention provides a basis for supplying protease to detergent formulations at a much lower cost per unit of enzyme activity. In addition, the enzyme of this invention has a higher optimum pH for protoeolytic activity; viz., pH 9.0-1l.0 as compared with an optimum pH range of pH 8.5-9.5 for a commercially available enzyme prepared from Bacillus subtilis. Furthermore, the enzyme of the present invention removes stains at least as well as, and in some instances, better than, the enzymes presently available on the market, and it has a greater solution stability in the pH range of 9.5-11.0. In general, it has been found, based on fermentation procedures set forth hereinafter, that the yield of alkaline protease obtained according to this invention is at least twice as high as is obtained from a hitherto high-yielding strain of Bacillus subtilis.

The Bacillus licheniformis A.T.C.C. No. 21424 of this invention is characterized by the following reactions:

Gelatin hydrolysis-positive Starch hydrolysis-positive Fermentation tests:

Sucroseacid production in 24 hours Xylose-acid production in 72 hours Arabinoseacid production in 72 hours Salicin-acid production in 96 hours Lactosenegative Glucose-nitrate agar slants-abundant growth Nitrites from nitratespositive Sporangia-very little swelling Cells displayed mobility and non-encapsulation. All of these tests are typical of both Bacillus subtilis and Bacillus licheniformis. The following additional tests were performed to confirm classification as B. licheniformis.

Growth on various carbon sources-Each of the three organisms were incubated in a modified Kosers citrate medium for ten days at 37 C. The media contained 0.2% of either sodium citrate, sodium acetate or sodium propionate. In addition, the media contained 1% NaCl, 0.1% (N I-I HPO 0.05 KH PO 0.02% MgSO .7H O, 1.5% agar and 0.0008% phenol red. The medium was adjusted to pH 7.0. At the end of the incubation period, growth was evaluated by changes in the color indicator from yellow to red.

B. licheniformic metabolizes all three organic salts while B. subtilis utilizes only citrate. Utilization of the anions by the organism released Na which in turn raised the pH of the medium and changed the indicator from yellow to red. Thus, the B. subtilis displayed red coloration on the citrate slants but no change on the acetate and propionate media. The other two organisms exhibited red coloration on all of the slants.

Control organisms used were B. subtilis A.T.C.C. No. 15841 and B. licheniformis A.T.C.C. NO. 12759. 'Both of these organisms as well as applicants B. licheniformz's A.T.C.C. No. 21424 were maintained on Trypticase Soy Agar slants (B.B.L. Labs) at 4 C. prior to use. All inoculations made in the tests were made directly from the slants.

Growth under anaerobiosis.-Each of the three organisms described above was inoculated into tubes of Trypticase Soy broth containing 0.095% thioglycolate and 0.005 cystine. In addition, stabs were made of each organism utilizing Anaerobic Agar without Dextrose or Eh. Indicator (B.B.L. Labs). This agar contained 20 g. Trypticase, a peptone derived from casein by pancreatic digestion, 5 g. NaCl, 15 g. Agar, 2 g. Na thioglycolate and 1 g. Na Formaldehyde sulfoxylate per liter. After incubation for 10 days at 37 C., the cultures were observed for growth.

Under these conditions, each organism grew in the thioglycolate broth. However, the two strains of B. licheniform-is exhibited more growth after 7 days of incubation than the B. subtilis. Similar observations were made in the stab tests. Furthermore, the B. licheniformis strains displayed the ability to grow to the bottom of the stab, whereas growth appeared only in the upper part of the B. subtilis stab. This indicates that the B. licheniformis strains are more facultatively anaerobic than B. subtilis.

Growth at 56 C.-Each 0f the 3 microorganisms was inoculated on Trypticase Soy Agar slants and incubated at 56 C. for 18 hours. At the end of the incubation period, the cultures were observed for growth.

Under these conditions, the two strains of Bacillus licheniformis, A.T.C.C. No. 12759 and A.T.C.C. No. 21424, exhibited abundant growth, whereas no growth was observed with Bacillus subtilis, A.T.C.C. No. 15841.

The culture medium used consists of from 1% to 4% protein, from 1% to 12% glucose, and such mineral supplements as are necessary to insure proper growth of the microorganism. Soybean meal is a preferred protein raw material. Other protein substrates, such as casein, Pharmamedia, a cottonseed-derived protein material, and Proflo, a partially defatted cooked cottonseed flour, both sold by Traders Protein Div. of Traders Oil Mill Co., Fort Worth, Tex.

While glucose is preferred as a carbon source, corn meal and dextrin may be used. Commercial products such as Cerelose, a dextrose sold by Corn Products Company and having a high level of glucose, can also be used. The mineral salts found useful with the preferred culture medium for use according to this invention include MnCl MgCl CaCO CaCl NaH2PO4, Na HPO and ZnCl Maximum enzyme production is obtained when the medium is initially buffered at pH- 7.6. Yields became progressively less as the pH is varied in either direction from this value, and at pH values below 7 or above 8, the yield becomes unacceptably low.

Nearly all of the enzyme activity produced during incubation of the oragnism in the culture medium is found in the supernatant liquid after centrifugation. The enzyme is recovered from solution by any of the standard procedures known in the art.

In a preferred procedure which provides a high yield, a calcium salt, such as calcium acetate, is added to the culture medium before centrifugation. Insoluble calcium phosphates are formed which, in turn, absorb a gel-like material and both are removed by the centrifugation. The clear supernatant liquid, in this case, contains 98% of the total enzymatic activity in the culture medium.

The enzyme may be recovered from solution by adding one-third volume of acetone at 2 C. to the clear supernatant. The resulting precipitate is removed by centrifugation and discarded. Additional acetone is added until 75% saturation of the fluid is obtained. The suspension is stored at 20 C. until precipitation is complete (about three hours). The precipitate is collected and dried to yield a powder.

Comparisons of the enzyme prepared with B. licheniformis A.T.C.C. No. 21424 of this invention with alkaline protease prepared from B. subtillis strains used to make commercially available alkaline protease on an equal activity basis have shown a number of advantages for the enzyme of this invention. In determining the optimum pH for caseinolytic activity, applicants enzyme shows greater activity over the pH range t 9.5-11.

Heat inactivation characteristics were also compared. Both enzymes were incubated at various temperatures for minutes and then quickly cooled in ice water. The residual activity was assayed with casein at pH 9.5. At incubation temperatures below 40 C., both enzymes were stable. At 60 C., the enzyme from B. subtz'lis was'completely inactivated, whereas applicants enzyme retained 15% of its original activity. Under the same conditions at 60 C. but with the additional presence of 10 M calcium ions, applicants enzyme retained 30% of the proteolytic activity while the B. subtillis enzyme retained only Both enzymes displayed an optimum temperature for caseinolytic activity at 55 C. at pH 7.0. In the presence of calcium ions the optimum temperature shifted to 60 C.

The alkaline protease of the present invention is used as an active ingredient in enzyme detergent compositions. The enzyme may be in powder or liquid form, and may be diluted with other ingredients. It is customary to dilute enzyme powder, as obtained by recovery from fermentation processes, with a compatable diluent such as sodium sulfate. Alkaline protease of this invention is used in an enzyme detergent composition at a level varying from 0.02% to 0.1% of the powder. When the enzyme powder is diluted approximately fivefold with sodium sulfate, it has an activity of about 1300 proteolytic units/ mg.

In addition to the enzyme detergents typically contain detergent actives. Various surface active agents known to the art can be used as detergent actives according to this invention, including anionic, nonionic, zwitterionic, ampholytic detergent compounds and mixtures thereof.

Anionic detergent compositions which can be used in the compositions of this invention include both soap and nonsoap detergent compounds. Examples of suitable soaps are the sodium, potassium, ammonium and alkylolammo nium salts of higher fatty acids (C -C The sodium or potassium salts of the mixtures of fatty acids derived from coconut oil and tallow, i.e., sodium or potassium tallow and coconut soap, are useful. Examples of anionic organic non-soap detergent compounds are the water soluble salts, alkali metal salts, of organic sulfuric reaction products having in their molecular structure an alkyl radical taining [from about 8 to about 22 carbon atoms and a radical selected from the group consisting of sulfonic acid and sulfuric acid ester radicals. (Included in the term alkyl is the alkyl portion of higher acyl radicals.)

Examples of the synthetic detergents which form a part of the compositions of the present invention are the sodium or potassium alkyl sulfates especially those obtained by sulfating the higher alcohols (Cy-C1 carbon atoms) produced by reducing the glycerides of tallow or coconut oil; sodium or potassium alkyl benzenesulfonates, such as are described in United States Letters Patents No. 2,220,099 and No. 2,477,383 in which the alkyl group contains from about 9 to about 15 carbon atoms; other examples of alkali metal alkylbenzene sulfonates are those in which the alkyl radical is a straight chain aliphatic radical containing from about 10 to about 20 carbon atoms for instance, Z-phenyl-dodecanesulfonate and 3-phenyl-dodecanesulfonate; sodium alkyl glyceryl ether sulfonates, especially those ethers of the higher alcohols derived from tallow and coconut oil; sodium coconut oil fatty acid monoglyceride sulfates and sulfonates; sodium or potassium salts of sulfuric acid esters of the reaction product of one mole of a higher fatty alcohol (e.g., tallow or coconut oil alcohols) and about 1 to 6 moles of ethylene oxide; sodium or potassium slats of alkylphenol ethylene oxide ether sulfate with about 1 to about 10 units of ethylene oxide per molecule and in which the alkyl radicals contain about 9 to about 12 carbon atoms; the reaction product of fatty acids esterified withisethionic acid and neutralized with sodium hydroxide where, for example, the fatty acids are derived from coconut oil; sodium or potassium salts of fatty acid amide of a methyl tapride in which the fatty acids, for example, are derived from coconut oil; and others known in the art, a number being set forth in United States Letters Patents Nos. 2,486,921, 2,486,922 and 2,396,278.

Nonionic synthetic detergents may be broadly defined as compounds aliphatic or alkylaromatic in nature which do not ionize in water solution. For example, a well known class of nonionic synthetic detergents is made available on the market under the trade name of Pluronic. These compounds are formed by condensing ethylene oxide with an hydrophobic base formed by the condensation of propylene oxide with propylene glycol. The hydrophobic portion of the molecule which, of course, exhibits Water insolubility has a molecular weight of from about 1,500 to 1,800. The addition of polyoxyethylene radicals to this hydrophobic portion tends to increase the water solubility of the molecule as a whole and the liquid character of the product is retained up to the point where polyoxyethylene content is about 50% of the total weight of the condensation product.

Other suitable nonionic synthetic detergents include:

(1) The polyethylene oxide condensates of alkyl phenols, e.g., the condensation products of alkyl phenols having an alkyl group containing from about 6 to 12 carbon atoms in either a straight chain or branched chain configuration with ethylene oxide, the said ethylene oxide being present in amounts equal to 10 to 25 moles of ethylene oxide per mole of alkyl phenol. The alkyl substituent in such compounds may be derived from polyrnerized propylene, diisobutylene, octane or nonene, for example.

(2) Those derived from the condensation of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediarnine. For example, compounds containing from about 40% to about polyoxyethylene by weight and whose average molecular weight is from about 5,000 to about 11,000 resulting from the reaction of ethylene oxide groups with a hydrophobic base constituted of the reaction product of ethylene diamine and excess propylene oxide, said hydrophobic base having a molecular weight of the order of 2,500 to 3,000, are satisfactory.

(3) The condensation product of aliphatic alcohols having from 8 to 18 carbon atoms, in either straight chain or branched chain configuration, with ethylene oxide, e.g., a coconut alcohol-ethylene oxide condensate having from 10 to 30 moles of ethylene oxide per mole of coconut alcohol, the coconut alcohol fraction having from 1 to 14 carbon atoms.

(4) Long chain tertiary amine oxides corresponding to the following general formula, R R R N+O, wherein R is an alkyl radical of from about 8 to 18 carbon atoms, and R and R are each methyl or ethyl radicals. The arrow in the formula is a conventional representation of a semi-polar bond. Examples of amine oxides suitable for use in this invention include dimethyldodecylamine oxide, dimethyloctylamine oxide, dimethyldecylamine oxide, dimethyltetradecylamine oxide, dimethylhexadecylamine oxide.

(5) Long chain tertiary phosphine oxides corresponding to the following formula, RRR"P- O, wherein R is an alkyl, alkenyl or monohydroxyalkyl radical ranging from to 18 carbon atoms in chain length and R and R" are each alkyl or monohydroxyalkyl groups containing from 1 to 3 carbon atoms. The arrow in the formula is a conventional representation of a semi-polar bond. Examples of suitable phosphine oxides are:

dimethyldodecylphosphine oxide, dimethyltetradecylphosphine oxide, ethylmethyltetradecylphosphine oxide, octyldimethylphosphine oxide, dimethylstearylphosphine oxide, cetylethylpropylphosphine oxide, diethyldodecylphosphine oxide, diethyltetradecylphosphine oxide, bis(hydroxymethyl)dodecylphosphine oxide, bis (Z-hydroxyethyl dodecylphosphine oxide, 2-hydroxypropylmethyltetradecylphosphine oxide, dimethyloleylphosphine oxide, and dimethyl-Z-hydroxydodecylphosphine oxide.

(6) Dialkyl snlfoxides corresponding to the following formula, RRS O, wherein R is an alkyl, alkenyl, betaor gamma-monohydroxyalkyl radical containing one or two other oxygen atoms in the chain, the R groups ranging from 10 to 18 carbon atoms in chain length, and wherein R' is methyl or ethyl. Examples of suitable sulfoxide compounds are:

dodecylmethyl sulfoxide,

tetradecylmethyl sulfoxide, 3-hydroxytridecylmethyl sulfoxide, 2-hydroxydodecylmethyl sulfoxide, 3hydroXy-4-decoxybutylmethyl sulfoxide, 3-hydroxy-4-dodecoxybutylmethyl sulfoxide, 2-hydroxy-3-decoxypropylmethyl sulfoxide, 2-hydroxy-3-dodecoxypropylmethyl sulfoxide, dodecylethyl sulfoxide, and Z-hydroxydodecylethyl sulfoxide.

The 3-hydroxy-4-decoxybutyl methyl sulfoxide has been found to be an especially effective detergent surfactant. An outstanding detergent composition contains this sulfoxide compound in combination with the polymaleate builder compound of this invention.

Ampholytic synthetic detergents can be broadly de scribed as derivatives of aliphatic secondary and tertiary one contains an anionic water solubilizing group. Examples of compounds falling within this definition are 3- (N,N-dimethyl N hexadecylammonio)-propane-1-sulfonate and 3-(1N, N-dimethyl N hexadecylammonio)-2- hydroxypropane 1- sulfonate which are especially preferred for their excellent cool water detergency characteristics.

The anionic, nonionic, ampholytic and zwitterionic detergent surfactants mentioned above can be used singly or in combination in the practice of the present invention. The above examples are merely specific illustrations of the numerous detergents which can find application within the scope of this invention.

The foregoing organic detergent surfactant compounds can be formulated into any of the several commercially desirable composition forms, for example, granular, flake, liquid and tablet forms.

These detergents may also optionally contain either organic or inorganic detergency builders such as sodium tripolyphosphate, tetra sodium pyrophosphate, tetra potassium pyrophosphate, potassium tripolyphosphate, sodium hexametaphosphate, sodium nitrilotriacetate and the sodium salt of ethylene diamine tetraacetic acid.

Care must be taken to insure that the detergents of this invention do not contain agents which adversely affect or are adversely affected by the enzyme composition. For example, hypochloride bleaching agents must not be used, because they completely inactivate the enzyme. The so-called oxy bleaches may also exhibit an adverse effect. Furthermore, other protein and protein-like materials generally should not be added to these detergent compositions as they may be attacked by the alkaline protease of this invention.

EXAMPLE 1 A medium comprising 6% glucose, 2% soybean meal, 0.04% CaCI .02% MgCl in 0.1 M

buffer at pH 7.6 was prepared. The glucose was provided as a 50% solution, autoclaved separately. The balance of the medium is also autoclaved separately and the two portions combined under aseptic conditions.

An inoculum was prepared by adding 5 ml. of sterile distilled water to a 24-hour-old Trypticase Soy slant of Bacillus liclzeniformis, A.T.C.C. No. 21424, [Trypticase Soy Agar (BBL) has the following composition: Trypticase, a peptone derived from casein by pancreatic digestion, 15 grams; Phytone, a papian digest of soya meal, 5 grams; NaCl, 5 grams; and Agar, 15 grams, in 1 liter of water] that had been incubated for 24 hours at 37 C., and a cell suspension was prepared. The suspension was then introduced aseptically into a 250 ml. Erlenmeyer flask containing 50 ml. of the autoclaved medium described above. The flask was then shaken on a reciprocal shaker at r.p.m. for 18 hours at 37 C. At the end of the incubation period, a 10% inoculum (5 m1.) from the inoculum flask was introduced into fermentation flasks which contained 50 ml. of the autoclaved medium described above.

The fermentation flasks were shaken on a reciprocal shaker at 175 r.p.m. and 37 C. Maximum protease production occurred after 48-72 hours or at a terminal pH of 7.2-7.4.

On completion of the fermentation period, a 0.15 M aqueous solution of calcium acetate was added to the culture medium. The calcium salt addition caused the formation of insoluble calcium phosphates which, in turn, absorbed a gel-like substance in the medium. The insoluble compounds were then removed from the medium by centrifugation at 4 C. The clear supernatant contained approximately 98% of the total enzyme activity present in the culture medium.

Additional purification was carried out by adding onethird volume of acetone (25% saturation) to the clear supernatant at 2 C. The resulting precipitate was removed by centrifugation and discarded. Additional acetone was added until a 75% saturation was obtained. The resulting suspension was stored at 20 C. for approximately three hours until precipitation was complete. The precipitate was collected and subsequently dried, either by acetone drying or freeze-drying. Yields ranged from 800 to 1100 mg. of dried enzyme powder per liter of crude supernatant.

The strength of the enzyme preparations was assayed by the following assay based on digestion of casein.

Assay Add 1.9 ml. of 0.05 M Na boratel.0 N NaOH buffer (pH 9.5) to 1.0 ml. of 2% casein (Hammersten) solution also made with the same borate buffer, and equilibrate at 37" C. Then add 0.1 ml. of the enzyme sample, appropriately diluted, to the casein-bulfer mixture. After a lO-minute digestion at 37 C., 3.0 ml. of trichloroacetic acid was added and incubation was continued at 37 C. for additional 30 minutes to insure complete precipitation of the undigested casein. The precipitate was removed by filtration (Whatman No. 2 paper) and the absorbency of the clear filtrate was read in a spectrophotometer at 280 mu against a blank. The blank was prepared similarly, except that 3.0 ml. of 10% TCA was first added to the casein 1.9 ml. buffer mixture, and the enzyme sample (0.1 ml.) was added subsequently. One proteolytic unit, as the term is used herein and in the appended claims is arbitrarily defined as the amount of enzyme producing an increase in absorbancy (optical density) of 1.0 at 280 mu in ten minutes at 37 C. Eight hundred eighty-eight proteolytic units of this invention equals 1 Anson Unit.

EXAMPLE II PROTEASE PRODUCTION WITH VARIOUS CONCENTRA- TIONS OF SOYBEAN MEAL AND GLUCOSE Media:

Soybean meal (percent) 1 1 Glucose (percent) 1 2 3 4 6 Mg. niltrogen/ml. medium: Soybean e 2. 60 Total reducing sugars as mg. glucose/ml. medium:

Soybean meal--- Glucose Carbon: nitrogen r Proteolytic units/m1. medium at maximum production 1. 08 la s EXAMPLE III A medium comprising 3% glucose, 1% soybean meal, 0.04% CaCl 0.2% MgCl in 0.1 M

buffer at pH 7.6 was prepared. An inoculum was prepared as described in Example I from a 24-hour old Tryticase Soy Agar slant of Bacillus licheniformis A.T.T.C. No. 21424. 100 ml. of inoculum was introduced into one liter of the medium described above, and the flask was shaken in a reciprocal shaker at 175 r.p.m. and at 37 C. Respective samples were removed from the flask after 24, 48 and 72 hours, and the total bacterial count, the spore count, and the activity of the protease enzyme in the sample were determined after each of these time periods. The activity was determined in glycine units (G.U.). For purposes of comparison with the Proteolytic units described above, 1 proteolytic unit equals 846 glycine units. The results of the determinations were given in the following table:

TABLE 1.CULTURE 0F STRAIN and there appeared to be 50% sporulation after 72 hours. The proteolytic activity reached a maximum of 20,700 G.U./ml. after 48 hours. This represents an excellent yield. A high-yielding strain of Bacillus subtilis provided a maximum of 13,000 G.U./ml. after 40 hours using the same conditions.

The activity of the enzyme is expressed in glycine units per ml. or per mg. The enzyme was isolated from the culture broth after the indicated incubation time by cooling the liquid to 5 C., and centrifuging it at 17,300 g.350 ml./minute in a Sorvall cooled centrifuge type RC 2 B equipped with a Szent Gyorgiy continuous flow system. The broth was then mixed with 2 vol. acetone (20 C.) and after one hour the precipitate was centrifuged (6870 X g. 350 ml./ min). The precipitate was resuspended in about 300 ml. of a water-acetone mixture (1 vol./2 vol.). The slurry was filtered on a Buchner funnel and the precipitate was dried by adding more cold acetone.

The results obtained during isolation of the protease from five of the one-liter flasks are given in the following table:

TABLE 2 Volume broth (1) 5 Activity before working up (G.U./ml.) 17,300 Harvest (g.) 104 Activity of powder (G.U./ml.) 675 Yield in percent of initial activity 82 The losses during working up are very low. The yield of 82% compares favorably with a usual yield of 50% with most strains of B. subtilis.

EXAMPLE IV The enzyme preparation made in accordance with Example I is used as an active ingredient in a detergent composition having the following formula:

Percent Sodium alkyl aryl sulfonate 8.2 Sodium fatty alcohol sulfate 8.2 C fatty acid amide 1.5

Alkaline protease; dried enzyme powder prepared in process of Example I, diluted five-fold with sodium sulfate 2 Sodium tripolyphosphate 50 Sodium silicate 4.8 Sodium sulfate 13.4 Perfume .15 Miscellaneous 2.1

Water Balance EXAMPLE V The protease prepared in accordance with Example III is used as an active ingredient in the following detergent composition:

Percent C -C fatty alcohol 10 ED. condensate 2.7 Sodium alkyl aryl sulfonate 12.6

Miscellaneous 1:7

I claim:

1. A method for producing an alkaline protease which comprises the steps of introducing a culture of Bacillus licheniformis A.T.C.C. No. 21424 into a culture medium, allowing fermentation to take place until the culture medium contains a high level of alkaline protease, and recovering the accumulated alkaline protease.

2. A method for the production of alkaline protease which comprises the steps of growing Bacillus licheniformis A.T.C.C. No. 21424 under aerobic conditions in an aqueous medium having a pH between 7 and 8 and containing a carbon source, a protein and traces of min eral salts at a temperature within the range of 35 C. to 40 C. for a period of time from 48 to 96 hours, and thereafter recovering the enzyme from the culture medium.

3. A method for producing an alkaline protease in accordance with claim 2, wherein the culture medium comprises 3%-6% glucose, 1%-2% soybean meal, 0.04% CaCl and 0.02% MgCl in a 0.1 molar aqueous buffer at pH in the range of 7 to 8, the percentages being by weight.

4. A method for producing an alkaline protease in accordance with claim 2, wherein the culture medium comprises 6% glucose, 2% soybean meal, 0.04% CaCl and 0.02% MgCl in a 0.1 molar Na HPO =NaH PO aqueous buffer at a pH of 7.6, the percentages being by weight.

5. A method for producing an alkaline protease which comprises the steps of preparing an inoculum of Bacillus licheniformis A.T.C.C. No. 21424 in a culture medium comprising 6% glucose, 2% soybean meal, 0.04% CaCl and 0.02% MgCl in a 1 M Na HPO =NaH PO aqueous butter at a pH of 7.6, incubating the inoculum for about 24 hours at 37 C., inoculating fresh quantities of said culture medium with said inoculum, incubating the medium at 37 C. for 48 to 96 hours until the pH of medium reaches the range of 7.2-7.4, separating the alkaline protease from the medium and drying the protease.

6. The method of claim 5, wherein the alkaline protease is separated from the culture medium by (a) adding calcium acetate to a level of 0.15 M in the culture medium, (b) centrifuging at 4 C., and discarding the solids, (c) adding one-third volume of acetone to the supernatant at 2 C., (d) centrifuging and discarding the solids, (e) adding additional acetone until saturation is reached, (f) storing the suspension at -20 C. until precipitation is complete, and (g) recovering and drying the precipitate.

References Cited UNITED STATES PATENTS 3,451,935 6/1969 Roald et al. 195-68 X FOREIGN PATENTS 1,800,508 5/ 1969 Germany.

OTHER REFERENCES Bernlohr: Journal of Biological Chemistry, vol. 239, No. 2, pp. 538 to 543 (1964).

Hall et a1.: Archives of Biochemistry and Biophysics, vol. 114, pp. -153 (1966).

Hall: Dissertation Abstracts 27, 2277-B to 2278-B January (1967).

LIONEL M. SHAPIRO, Primary Examiner I; UNITED STATES PATENT OFFICE I CERTIFICATE OF CORRECTION Datent NC. 3, 748,233 Dated July 24, 1973 Ihventor(g) John P. Viccaro It is certified that error appears in the aboveidentified patent and that said Letters Batent are hereby corrected as shown below:

Column 8, line 68, "Example It" should read -Example III-P.

Signed and sealed this 1st day of January 1974.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. RENE D. TEGTMEYER Attesting. Officer Acting Commissioner of Patents 

